This invention relates to a mammalian cDNA which encodes a human aquaporin-8 variant and to the use of the cDNA and the encoded protein in the diagnosis and treatment of pancreatic disorders, particularly type I diabetes.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Aquaporins are ubiquitous water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins. Aquaporins contain sites which prevent ions from passing through the channel but allow water and certain other solutes such as urea and glycerol to pass through. Aquaporins are characterized by six membrane-spanning domains and five hydrophilic connecting loops containing two highly conserved asparagine-proline-alanine (NPA) amino acid motifs in the two halves of the protein. In mammals, aquaporins are found in exocrine glands such as the salivary glands and pancreas and in water-permeable epithelia of the lens, liver, testis, kidney, and colon. Aquaporin-8 (AQP8), discovered in rat pancreas and liver (Koyama et al. (1997) J Biol Chem 272:30329-30333), is highly homologous to plant xcex3- and xcex1-tonoplast intrinsic proteins, also members of the MIP family. Similar to that of the plant aquaporin, xcex3-TIP, AQP8 has a longer N terminus and a shorter C terminus compared with those of other mammalian aquaporins (Koyama, supra). AQP8 does not allow transmembrane transport of urea or glycerol. AQP8 expression is strong in glandular lobes of the pancreas and parenchymal cells of the liver, testis and seminiferous tubules, and weak in acinal cells in the salivary gland and absorptive columnar epithelial cell layer of the colon.
The discovery of a mammalian, cDNA encoding aquaporin-8 variant satisfies a need in the art by providing compositions which are useful in the diagnosis and treatment of pancreatic disorders, particularly type I diabetes.
The invention is based on the discovery of a mammalian cDNA which encodes a mammalian aquaporin-8 variant (AQP8V) which is useful in the diagnosis and treatment of pancreatic disorders, particularly type I diabetes.
The invention provides an isolated mammalian cDNA or a fragment thereof encoding a mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having 75% identity to the amino acid sequence of SEQ ID NO:1, an antigenic epitope of SEQ ID NO:1, an oligopeptide of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides an isolated mammalian cDNA or the complement thereof selected from the group consisting of a nucleic acid sequence of SEQ ID NO:2, a variant having at least 81% identity to the nucleic acid sequence of SEQ ID NO:2, a fragment of SEQ ID NOs:3-11, an oligonucleotide of SEQ ID NOs:2-11. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA ,or the complement of the cDNA, encoding AQP8V. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make AQP8V. The invention still further provides a transgenic cell line or organism comprising the vector containing the cDNA encoding AQP8V. The invention additionally provides a mammalian fragment or the complement thereof selected from the group consisting of SEQ ID NOs:3-11. In one aspect, the invention provides a substrate containing at least one of these fragments. In a second aspect, the invention provides a probe comprising the fragment which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose type I diabetes. In another aspect, the cDNA or a fragment or a complement thereof may comprise an element on an array.
The invention additionally provides a method for using a cDNA or a fragment or a complement thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, enhancers, repressors, and regulatory molecules.
The invention provides a purified mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, an oligopeptide of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides a composition comprising the purified protein or a portion thereof in conjunction with a pharmaceutical carrier. The invention further provides a method of using the AQP8V to treat a subject with type I diabetes comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with type I diabetes.
The invention provides a method of using a mammalian protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions that allow specific binding, dissociating the antibody from the bound-protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of type I diabetes.
The invention also provides a method of using a mammalian protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified antibodies.
The invention provides a purified antibody which binds specifically to AQP8V. The invention also provides a method of using an antibody to diagnose type I diabetes comprising combining the antibody comparing the quantity of bound antibody to known standards, thereby establishing the presence of type I diabetes. The invention further provides a method of using an antibody to treat type I diabetes comprising administering to a patient in need of such treatment a pharmaceutical composition comprising the purified antibody.
The invention provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:2-11, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.